Complementarity-determining region clustering may cause CAR-T cell dysfunction

Chimeric antigen receptor (CAR)-T cell therapy is rapidly advancing as cancer treatment, however, designing an optimal CAR remains challenging. A single-chain variable fragment (scFv) is generally used as CAR targeting moiety, wherein the complementarity-determining regions (CDRs) define its specificity. We report here that the CDR loops can cause CAR clustering, leading to antigen-independent tonic signalling and subsequent CAR-T cell dysfunction. We show via CARs incorporating scFvs with identical framework and varying CDR sequences that CARs may cluster on the T cell surface, which leads to antigen-independent CAR-T cell activation, characterized by increased cell size and interferon (IFN)-γ secretion. This results in CAR-T cell exhaustion, activation-induced cell death and reduced responsiveness to target-antigen-expressing tumour cells. CDR mutagenesis confirms that the CAR-clustering is mediated by CDR-loops. In summary, antigen-independent tonic signalling can be induced by CDR-mediated CAR clustering, which could not be predicted from the scFv sequences, but could be tested for by evaluating the activity of unstimulated CAR-T cells.

1. CAR expression on the surface of T cells should be shown for all 5 CARs associated with Figure  1. Is there a difference in CAR surface expression in these different CARs? 2. While the % binding of the 5 CARs to U87 was fairly similar, it is clear that MFI is lowest likely in E, suggesting that affinity measures to hIL13Ra2 and to cells expressing IL13Ra2 may be different. Please explain in the context of tonic signaling. If affinity is different between these 5 CARs, it is difficult to interpret the differences in on-target activity as well as tonic signaling. 3. While the authors show scFv(B) as a soluble form being able to bind to IL13Ra2 molecules on tumor cells, they do not show antigen-specific binding in the CAR form; the authors should stain CAR(B) T cells with recombinant soluble IL13Ra2, and compared with CAR(E). This would also confirm that lack of activity of CAR(B) is due to tonic signaling, and not just due to lack of binding to target. 4. Since there seems to be a lack of IFNg and T cell activation of the 5 scFvs against antigennegative tumors cells, but significant activation/exhaustion markers shown in Figure 2 in resting CAR(B) T cells, it would be prudent to justify the discrepancy and explain in more detail the differences between these two experimental conditions. 5. Critical data is missing here in the mutant CAR(B) constructs, specifically T cell cytotoxicity, T cell expansion/proliferation, and in vivo therapy activity in comparison to WT CAR(B) and CAR(E). 6. Additionally, affinity measures of these variants should be performed to compare to CAR(B) and specifically confirm that all functional readouts in Figure 4 is not due to loss of affinity of the CAR variants. Otherwise, the diminished cell size, IFNg production, and activation/exhaustion markers may be due to disruption/reduction of antigen binding and/or loss of CAR T cell activity.
Additional comments: 1. For all data, please include timepoints associated with analysis for each. Figure 1R should be presented in log10 scale, and consider placing survival after flux imaging and quantification. 3. IL13Ra2 expression flow plots (associated with MFI in Figure S2 should be shown comparing U87 and U343 lines associated with Figure 1.

Flux in
Reviewer #2 (Remarks to the Author): In this manuscript, Sarén et al. report the generation of CAR-T cells derived from five different scFv that only differed in the CDR loop sequences. They demonstrate that CDR sequences have an impact on CAR expression and can drive tonic signaling, which may result in impaired anti-tumor effects. The manuscript is well-written, and the experiments are well-designed, including the necessary controls. Overall, the quality of this study is high, and the conclusions are largely supported by the data. However, while the findings in this work are interesting, there are some significant weaknesses that raise many questions. The authors need to address the following concerns in order to enhance the quality of this study: This work addresses an immunologically relevant issue to understand the behavior of different CAR constructs. However, there is enough evidence in the field showing that the combination of certain scFv and costimulatory domains can drive tonic signaling. The main question to resolve is how can we design CARs whose configuration do not induce tonic signaling. Is there a specific amino acid combination responsible for CAR oligomerization? Can we predict CAR oligomerization based on computational modelling techniques to be used in silico? Can we mutate the CDR to prevent CARmediated tonic signaling while improving CAR-mediated killing? Would the combination of the scFv B with a different costimulatory domain (i.e. CD28) result in less tonic signaling? The lack of solutions to the issues highlighted in this manuscript render their results incomplete.
Tonic signaling is dependent on the levels of CAR expression. Flow cytometry plots showing CAR expression should be included in Figure 1 for scFv A-E and in Figure 4 for all mutants. Also, the authors state in the discussion that CAR expression on CAR(B) T cells is reduced over time. These results should be included in the main manuscript.
A functional characterization of the CAR(B) mutants upon antigen recognition should be included.
Why did the authors choose to characterize tonic signaling only on CAR(B) T cells? Do the other scFv also induce tonic signaling? Rescuing CAR(B)-T cell efficacy by mutating the CDR may be challenging, but could this strategy be used to improve the in-vivo antitumor efficacy of CAR(C) and CAR(D), which showed promising in vitro efficacy?
Finally, the authors state that they have identified a candidate CAR-T that warrant clinical translation for the treatment of recurrent glioblastoma. However, they have only tested the candidate CAR-T cells in one in vivo experiment. The anti-tumor efficacy of CAR(E)-T cells should be tested in an additional animal model.

Minor comments:
-To which treatment group do the animals shown in Figure 1S belong? -The authors stated: "we created a double mutant, with wild-type H2 and the most common L3 sequence ([B]-H2L3wt)"-What do authors mean by "the most common L3 sequence"? How did they choose mutations in CDR2 of the heavy chain and CDR3 of the light chain? -The in vivo antitumor efficacy of CAR-T C and D should be moved from supplementary data to The authors describe the generation of a set of 5 human IL13Rα2-specific Fcv molecules that did not cross-react with human IL13Rα1.  [E]. They find that mutation of some of these residues prevents aggregation of the construct and rescues the tonic signalling. Finally, they show that tonic signalling is mediated by the cytoplasmic tail in the aggregated forms of the receptors by generating tail-less "decoy" receptors, showing tail-less CAR [B] does not induce tonic signalling.
In general the quality of the work is very good, but I am left wondering what the specific message of the paper is. It seems to have two separate parts, the first being a description of a candidate CAR for glioblastoma treatment and the second an in depth analysis of why some of the candidate CAR constructs failed to work. Perhaps some better integration of the two parts in the discussion would help me as a reader understand whether this study is primarily describing a CAR construct that will be taken further into clinical trials, or whether it is meant to be instructive for others developing CAR therapies and describing mechanisms that lead to tonic signalling in constructs.
Some specific comments: - Figure 2B: It doesn't look like PC3 is needed to separate the conditions (unless there was a difference in PC3 between Mock and CAR[E]-T). The figure as plotted also makes it impossible to distinguish differences in PC3. The authors could consider plotting the data on a 2D PC1 vs PC2 with the size of points scaled to values of PC3, or have 3 plots of PC1 vs PC2, PC1 vs PC3 and PC2 vs PC3 or perhaps just leaving PC3 out and plotting PC1 vs PC2 and mentioning that PC3 did not aid in separating categories.
- Figure 4: To tie together the concept that aggregation induces signalling through the cytoplasmic tail, data showing that CAR[B]d aggregates like CAR [B] (like the data shown in panel E and quantified in panel F) is required. This would also confirm that aggregation is not mediated by interactions between signalling molecules associated with the cytoplasmic tails of receptors activated by some potential conformational mechanism.
-As the authors note in the discussion, it is well known that CDR loop sequences can induce aggregation, and that aggregation of CARs induces tonic signalling. Most published work has focussed on the contribution of the framework and hinge regions of CARs to aggregation and tonic signalling, and as such this is one of the few to follow up candidate CARs with high tonic signalling and show that the CDR loops in the scFv are responsible. Most others would assume this is the case and discard the candidate. The bigger question is whether promising scFv candidates can be rescued. The authors used alanine mutants or substitutions of sequences ([B]-H3L2wt) to show that tonic signalling can be reversed, but determining to what degree this has affected binding to antigen or how effectively CAR-T cells with these constructs can be activated by cells expressing the target antigen would be useful.

Response to specific questions to each reviewer
Reviewer #1 (Remarks to the Author): In this manuscript by Saren et al., the authors identified and evaluate several IL13Ra2-targeting CAR constructs with varying scFvs and their effector function in vitro and in vivo. They identified differential activity of these scFv-based CARs, and that one scFv (CAR(B)) had greater antigenindependent tonic signaling. Importantly, changes in sequence of CAR(B) in the CDR reduced clustering, tonic signaling by lower IFNg production and activation/exhaustion markers. However, several key studies are lacking in this well-executed and displayed work. Critical data on these mutant CAR(B) constructs, including cytotoxicity, T cell expansion/proliferation, and in vivo therapy are missing. These studies would be required to confirm that these changes in tonic signaling are not simply due to a lack of CAR T cell activity/targeting of IL13Ra2. Further, it is unclear whether this CDR-driven clustering phenomenon extends beyond IL13Ra2, and if not, this would limit the broad applicability of these findings to the CAR field. Nonetheless, the following could be address to improve the impact of this study: We greatly appreciate the reviewer's positive feedback. We agree with the reviewer that we did not show that CDR-driven clustering and subsequent antigen-independent tonic signaling is a general phenomenon extending beyond IL13Rα2-targeting CARs. Therefore, in the updated version of the manuscript we have demonstrated CDR-mediated clustering for an additional IL13Rα2-targeting CAR, namely CAR[A]-T cells in Supplementary Figure S8. Furthermore, we show CDR-mediated clustering can also be observed in CARs targeting CD44v6 in Supplementary Figures S9 and S10. We thereby strengthen that our findings have a broader applicability in the CAR-T cell field.  Figure S6A. We believe that the difference between surface expression between the 5 CARs is due to CAR stability with CAR[A] and CAR [B] being unstable leading to reduce surface expression and lower functionality. This is shown in the revised manuscript in the Results section (lines 119-125.
2. While the % binding of the 5 CARs to U87 was fairly similar, it is clear that MFI is lowest likely in E, suggesting that affinity measures to hIL13Ra2 and to cells expressing IL13Ra2 may be different. Please explain in the context of tonic signaling. If affinity is different between these 5 CARs, it is difficult to interpret the differences in on-target activity as well as tonic signaling.
We thank the reviewer for allowing us to explain this further. As the tonic signaling evaluated in this manuscript is an antigen-independent phenomenon (performed on resting CAR-T cells that have not seen the cognate CAR target antigen) the affinity of the CARs to the target antigen will most likely not influence antigen-independent tonic signaling. We agree that the difference in affinity between the 5 CARs could have an impact in terms of on-target activity. However, published literature on this have shown that high affinity CARs have normally better function in vitro than low affinity CARs (PMID: 26330166, PMID: 29085043). In our data, scFv[A]-scFv [C] have the highest affinity to IL13Rα2 ( Figure 1B) which also reflects the binding of CAR[A]-[C] to IL13Rα2 (Supplementary Figure S2A). As expected, CAR[C]-T is highly functional, however, CAR[A]-T and CAR[B]-T display poor functionality despite their high affinity. Furthermore, although CAR[C] has higher affinity than CAR [D] and CAR [E], CAR[E]-T performs better. As our data does not follow the common understanding of the field, we believe that affinity and target binding is not the major contributor to the results we observe but instead the CDR-mediated CAR clustering which results in antigenindependent activation and subsequent tonic signaling and CAR-T cell exhaustion.
3. While the authors show scFv(B) as a soluble form being able to bind to IL13Ra2 molecules on tumor cells, they do not show antigen-specific binding in the CAR form; the authors should stain CAR(B) T cells with recombinant soluble IL13Ra2, and compared with CAR(E). This would also confirm that lack of activity of CAR(B) is due to tonic signaling, and not just due to lack of binding to target.
We thank the reviewer for this comment. We have now performed the suggested experiment using recombinant soluble IL13Rα2 to show the binding of the 5 CARs to the receptor. The binding data for the 5 CARs is shown in Figure S2A  4. Since there seems to be a lack of IFNg and T cell activation of the 5 scFvs against antigen-negative tumors cells, but significant activation/exhaustion markers shown in Figure 2 in resting CAR(B) T cells, it would be prudent to justify the discrepancy and explain in more detail the differences between these two experimental conditions. We appreciate that the reviewer pointed this out and gave us the opportunity to explain this better. IFN-γ secretion from CAR-T cells into the co-culture supernatant of CAR-T cells together with antigen-negative tumor cells (Mel526) was evaluated by ELISA (Supplementary Figure S2K). In Figure 2 ( Figure 3 in the revised manuscript) the gene expression was assessed. Gene expression analysis is more sensitive than ELISA and thus explain why although activity might be evident at the gene expression level no IFN-γ can be detected by ELISA.
We would also like to take the opportunity to clarify the difference between the experimental settings used to obtain the different samples used for IFN-γ detection in the manuscript. CAR[A]-T cells and CAR[B]-T cells secrete IFN-γ into the culture supernatant even without antigen-stimulation (Fig. 2I).

Despite this, for CAR[A]-T cells and CAR[B]
-T cells, no IFN-γ was detected upon co-culture with antigen-negative (Mel526) cells (Fig. S2K) and very low levels of IFN-γ were detected after antigen stimulation (Fig. 1G-H; Fig. S2F-G). This can be explained as the assays were performed at different time points after T cell transduction. The co-culture experiment was set up at a later time point after T cell transduction and rapid expansion, and due to prolonged exposure to tonic signaling the ability to secrete IFN-γ was reduced. We have clarified this in the revised manuscript by adding at what time point after T cell transduction the experiments were performed in all Figure legends. 5. Critical data is missing here in the mutant CAR(B) constructs, specifically T cell cytotoxicity, T cell expansion/proliferation, and in vivo therapy activity in comparison to WT CAR(B) and CAR(E).
We thank the reviewer for this comment and we have performed new experiments to address this. We have extended the evaluation of antigen-independent tonic signaling of CAR[B] mutants in Figure 4. As suggested, we have also evaluated the response of the CAR[B] mutants to antigen stimulation (Supplementary Figure S7C-J). However, we would like to highlight that CAR [B] was mutated to confirm that the CDRs were responsible for CAR clustering and subsequent antigen-independent tonic signaling. Thus, we did not intend to rescue CAR[B] with kept affinity for the target antigen through mutation of the CDR loops as this certainly may impair binding and specificity to the target antigen. As expected, the non-tonically signaling [B]-H2L3wt did no longer bind to the target antigen.
[B]-L3 on the other hand, which displayed partially reduced tonic signaling, showed slightly better response upon antigen stimulation compared to CAR[B].
6. Additionally, affinity measures of these variants should be performed to compare to CAR(B) and specifically confirm that all functional readouts in Figure 4 is not due to loss of affinity of the CAR variants. Otherwise, the diminished cell size, IFNg production, and activation/exhaustion markers may be due to disruption/reduction of antigen binding and/or loss of CAR T cell activity.
We apologize for not being clear enough in our description of Figure 4. All studies with the CAR[B] mutants displayed in Figure 4 were performed without antigen-stimulation to show the effect of the different mutations on antigen-independent tonic signaling. We have now emphasized this both in the manuscript text and Figure 4 Legend regarding the experimental setting.
We thank the reviewer for pointing out the potential change in the binding for these mutants and have now evaluated the binding of the CAR[B] mutants to recombinant human IL13Rα2. It is presented in Supplementary Figure S7B.
Additional comments: 1. For all data, please include timepoints associated with analysis for each.
We thank the reviewer for pointing this out. We have now addressed this issue and added all time points at which the assays were performed in the figure legends. Figure 1R should be presented in log10 scale, and consider placing survival after flux imaging and quantification.

Flux in
We thank the reviewer for addressing this. As both Reviewer #3 and the editor think that we have 2 stories in our original manuscript, we have now placed the in vivo data in Supplementary Figure S3 and as suggested present the tumor growth in log10 scale. Figure S2 should be shown comparing U87 and U343 lines associated with In this manuscript, Sarén et al. report the generation of CAR-T cells derived from five different scFv that only differed in the CDR loop sequences. They demonstrate that CDR sequences have an impact on CAR expression and can drive tonic signaling, which may result in impaired anti-tumor effects. The manuscript is well-written, and the experiments are well-designed, including the necessary controls. Overall, the quality of this study is high, and the conclusions are largely supported by the data. However, while the findings in this work are interesting, there are some significant weaknesses that raise many questions. The authors need to address the following concerns in order to enhance the quality of this study:

IL13Ra2 expression flow plots (associated with MFI in
This work addresses an immunologically relevant issue to understand the behavior of different CAR constructs. However, there is enough evidence in the field showing that the combination of certain scFv and costimulatory domains can drive tonic signaling. The main question to resolve is how can we design CARs whose configuration do not induce tonic signaling. Is there a specific amino acid combination responsible for CAR oligomerization? Can we predict CAR oligomerization based on computational modelling techniques to be used in silico? Can we mutate the CDR to prevent CARmediated tonic signaling while improving CAR-mediated killing? Would the combination of the scFv B with a different costimulatory domain (i.e. CD28) result in less tonic signaling? The lack of solutions to the issues highlighted in this manuscript render their results incomplete.
We greatly appreciate the positive feedback from the reviewer. We agree that one of the major tasks in the field of antibody, scFv and CAR engineering is to predict and be able to avoid oligomerization and thereby be able to design a CAR devoid of clustering and subsequent tonic signaling. We have unfortunately not been able to resolve this issue in the current manuscript. The aim with our manuscript is to report on a new phenomenon: that CDR-mediated clustering, without any antigen stimulation, can cause tonic signal in CAR-T cells. So far, only the framework region of the scFv has been described associated with tonic signaling. In addition, to help scientists in selecting CARs to avoid tonic signaling, we also proposed that the activity of resting (not stimulated by the cognate antigen) CAR-T cells, e.g., enlarged cell size and background IFN-gamma secretion could be used as an easy method to exclude CARs that are prone to tonic signaling. This is important, as we suggest in the manuscript that CAR clustering and tonic signaling cannot be predicted at the scFv-level but must be evaluated at the CAR-T cell level. For example, the melting temperature (thermal stability) of scFv [B] is similar to that of the FMC63 clone used for construction of clinically approved CD19directed CAR-T cells. Yet CAR[B]-T cells are prone to tonic signaling while, CD19 CAR-T cells are not. We have further strengthened the importance of our findings to the CAR-T cell field by showing that CDR-mediated tonic signaling is a general phenomenon seen in CARs against another target then IL13Rα2, in this case against CD44v6 ( Supplementary Figures S9 and S10).
We also believe that changing the co-stimulatory domain from 4-1BB to e.g., CD28 would not rescue the tonic signaling as 4-1BB has been shown to result in lower tonic signaling compared to CD28 (doi:10.1038/nm.3838, PMID 25939063). Additionally, our data suggests the tonic signaling was caused by CDR-mediated scFv clustering, which further leads to signal transduction via the downstream co-stimulatory domain leading to non-responsive CAR-T cells.
Tonic signaling is dependent on the levels of CAR expression. Flow cytometry plots showing CAR expression should be included in Figure 1 for scFv A-E and in Figure 4 for all mutants. Also, the authors state in the discussion that CAR expression on CAR(B) T cells is reduced over time. These results should be included in the main manuscript.
We agree with the reviewer that the level of tonic signaling can vary with CAR expression and that for a CAR prone to clustering, higher CAR expression would likely lead to higher tonic signaling. For a CAR not prone to clustering, such as CAR[E], the expression level of CARs on the surface of T cells is retained over time while for a CAR prone to clustering, such as CAR [B], clustering will lead to aggregation and reduced level of CAR on the surface of the T cells over time. We have now added the requested data of expression level to the revised version of the manuscript, as main Figure  We thank the reviewer for pointing this out and have now performed a more detailed characterization of the CAR[B] mutants both without antigen stimulation and with antigen stimulation. These data can now be found in Figure 4 and Supplementary Figure S7. However, we would also like to highlight that the aim of creating the CAR[B] mutants was to confirm that the CDR loops caused CAR clustering and subsequent antigen-independent tonic signaling and determine which CDR loops were responsible for clustering in CAR [B]. In the revised manuscript we have performed mutation of another IL13Rα2directed CAR that was identified in our screen, CAR[A] (Supplementary Figure S8). In general, the data obtained from mutating the CDRs of CAR[A] corroborate the data already presented for CAR [B] in the first version of the manuscript. We have also assessed the binding of all mutants to recombinant IL13Rα2. In one case, when mutating CDR3 of the light chain for CAR [B], named [B]-L3, we observed reduced tonic signaling with maintained binding to IL13Rα2. This was however not the case when simultaneously mutating CDR3 of the light chain and CDR2 of the heavy chain for CAR [B], named [B]-H2L3wt, where tonic signaling was completely abolished but the binding to IL13Rα2 was at the same time lost. When mutating CAR[A], we found that it was mainly CDR3 of the heavy chain that was mediating clustering. When it was mutated, clustering was abolished but binding to IL13Rα2 was at the same time lost.
Why did the authors choose to characterize tonic signaling only on CAR(B) T cells? Do the other scFv also induce tonic signaling? Rescuing CAR(B)-T cell efficacy by mutating the CDR may be challenging, but could this strategy be used to improve the in-vivo antitumor efficacy of CAR(C) and CAR(D), which showed promising in vitro efficacy?
We thank the reviewer for bringing this up. The reasons why we chose to characterize CAR[B]-T cells further was that it was the most tonically signaling CAR among the 5 CARs and had the lowest cytotoxic potential against target cells. We agree with the reviewer that CAR[B]-T might be the most difficult CAR to rescue. However, we would like to emphasize that the intention with the mutation analysis was not to rescue the CAR-T functionality but to determine if the amino acids in the CDR regions were responsible for CAR clustering. This is also the reason we replaced corresponding CDRs using the nonpolar amino acid alanine, to minimize the side change interaction and thus reduce the likelihood for clustering. Indeed, the binding activity towards IL13Rα2 was affected by introducing mutations as presented in Supplementary Figure S7.
In the revised manuscript we also mutated CAR[A] to show that the CDRs were also causing tonic signaling in CAR[A]-T cells (Supplementary Figure S8). Clustering could be avoided by mutating certain amino acids but at the cost of compromising binding capacity. To further strengthen our findings, we also evaluated if CDR-mediated clustering was a general phenomenon existing beyond IL13Rα2. Importantly we found that CDR-mediated CAR-clustering of CD44v6-targeting CARs could induce antigen-independent tonic signaling ( Supplementary Figures S9 and S10) showing generalizability of our findings.

We have indications that CAR[C], CAR[D] and CAR[E]
-T cells display a low degree of tonic signaling as they have a somewhat larger cell size and secrete low levels of IFN-γ without antigenstimulation in comparison to Mock-T control. We believe that this level of tonic signaling might even be beneficial to lowering the activation threshold of these constructs without inducing dysfunction. A recent study has shown that a low level of tonic 4-1BB signaling is even beneficial for CAR T cells (doi:10.1038/s41591-021-01326-5, PMID: 33888899).
Finally, we got the comment from the editor that the paper had two parallel stories, tonic signaling which we consider the most important one for this manuscript and the therapeutic efficacy against glioblastoma in vivo with further clinical translation. Therefore, the in vivo anti-tumor efficacy part has been reduced in the revised manuscript.
Finally, the authors state that they have identified a candidate CAR-T that warrant clinical translation for the treatment of recurrent glioblastoma. However, they have only tested the candidate CAR-T cells in one in vivo experiment. The anti-tumor efficacy of CAR(E)-T cells should be tested in an additional animal model.
We agree that if the main focus of the paper would have been to promote CAR[E]-T cells for clinical translation an additional model would certainly be needed. However, following the suggestion of the editor and focusing our paper, we have formatted the manuscript in a different way, now mainly focusing on the tonic signaling part, while taken out the therapeutic part into Supplementary Figure S3 to make the story coherent. By downscaling the therapeutic part, the editor did not suggest us to add a second animal model (please read the editor's comment), but instead focus to the main story line. We hope that this is acceptable.
Minor comments: -To which treatment group do the animals shown in Figure 1S belong?
We have put the therapeutic data into Supplementary Figure S3 in the revised manuscript and made sure that it is clear which images belongs to which treatment group.
-The authors stated: "we created a double mutant, with wild-type H2 and the most common L3 sequence ([B]-H2L3wt)"-What do authors mean by "the most common L3 sequence"? How did they choose mutations in CDR2 of the heavy chain and CDR3 of the light chain?
We apologize for not explaining this properly and thank the reviewer for bringing this up. We used a human synthetic fragment library in our phage display library to obtain the single chain variable fragments. The particular CDR-L3 sequence we used in the mutant is the most frequently occurring CDR-L3 sequence within functional antibodies of the human IGKV1-39 germline that our library was based upon. For CDR-H2 the wild type H2 sequence is from the human IGHV3-23 germline, which the library was based on for the heavy chain, was used. We have now added additional sentences both in the result section (line 243) and materials and methods section (lines 392-393) of the revised manuscript to clarify this.
-The in vivo antitumor efficacy of CAR-T C and D should be moved from supplementary data to Figure 1.
To get the main message out, which is that certain CDR sequences or combination of CDR sequences can lead to CAR clustering, tonic signaling and CAR-T dysfunction we have instead moved out the therapeutic in vivo data from the main Figure into Supplementary Figure S3, in order to make the revised manuscript focused on the technical aspect and the role of the CDRs in CAR clustering.
Reviewer #3 (Remarks to the Author): The authors describe the generation of a set of 5 human IL13Rα2-specific Fcv molecules that did not cross-react with human IL13Rα1.  [E]. They find that mutation of some of these residues prevents aggregation of the construct and rescues the tonic signalling. Finally, they show that tonic signalling is mediated by the cytoplasmic tail in the aggregated forms of the receptors by generating tail-less "decoy" receptors, showing tail-less CAR [B] does not induce tonic signalling.
In general the quality of the work is very good, but I am left wondering what the specific message of the paper is. It seems to have two separate parts, the first being a description of a candidate CAR for glioblastoma treatment and the second an in depth analysis of why some of the candidate CAR constructs failed to work. Perhaps some better integration of the two parts in the discussion would help me as a reader understand whether this study is primarily describing a CAR construct that will be taken further into clinical trials, or whether it is meant to be instructive for others developing CAR therapies and describing mechanisms that lead to tonic signalling in constructs.
We appreciate the positive feedback from the reviewer. We agree that the paper was written in a way so that it looked like two separate parts, and we got the same comment from another reviewer and the editor. We have therefore now re-written it in a way to make the message of the paper clearer and only focus on the tonic signaling part. In light of this, we have also extended our evaluation of antigenindependent tonic signaling beyond IL13Rα2. In the revised manuscript we show that CDR-mediated tonic signaling is also observed in CARs directed against CD44v6 ( Supplementary Figures S9 and  S10).
8 Some specific comments: - Figure 2B: It doesn't look like PC3 is needed to separate the conditions (unless there was a difference in PC3 between Mock and CAR[E]-T). The figure as plotted also makes it impossible to distinguish differences in PC3. The authors could consider plotting the data on a 2D PC1 vs PC2 with the size of points scaled to values of PC3, or have 3 plots of PC1 vs PC2, PC1 vs PC3 and PC2 vs PC3 or perhaps just leaving PC3 out and plotting PC1 vs PC2 and mentioning that PC3 did not aid in separating categories.
We thank the reviewer for pointing this out and we agree with the comment. We have now changed this figure into a 2D PCA instead ( Figure 3B in the revised manuscript).
- Figure 4: To tie together the concept that aggregation induces signalling through the cytoplasmic tail, data showing that CAR[B]d aggregates like CAR [B] (like the data shown in panel E and quantified in panel F) is required. This would also confirm that aggregation is not mediated by interactions between signalling molecules associated with the cytoplasmic tails of receptors activated by some potential conformational mechanism.
We thank the reviewer for this valid point. We have now studied the aggregation of CAR[B]d and compared it to CAR[E]d and seen that CAR[B]d still aggregates. This data has now been added into Supplementary Figure S5B in the revised manuscript.
-As the authors note in the discussion, it is well known that CDR loop sequences can induce aggregation, and that aggregation of CARs induces tonic signalling. Most published work has focussed on the contribution of the framework and hinge regions of CARs to aggregation and tonic signalling, and as such this is one of the few to follow up candidate CARs with high tonic signalling and show that the CDR loops in the scFv are responsible. Most others would assume this is the case and discard the candidate. The bigger question is whether promising scFv candidates can be rescued. The authors used alanine mutants or substitutions of sequences ([B]-H3L2wt) to show that tonic signalling can be reversed, but determining to what degree this has affected binding to antigen or how effectively CAR-T cells with these constructs can be activated by cells expressing the target antigen would be useful.
We agree that to rescue promising scFv candidates is one of the biggest challenges in the field of antibody and CAR-T engineering. A universal solution to this would be remarkably important. Unfortunately, we cannot provide such solution with our findings. Importantly, our data clearly suggest that screening of scFvs as recombinant proteins is not sufficient but that the scFvs need to be screened in the CAR format to determine if they cause tonic signaling. In the revised manuscript, we strengthened our findings by proposing to use cell size and autonomous IFN-gamma secretion of unstimulated CAR-T cells as easy method for CAR candidates screening.
We performed alanine substitution with the aim to confirm that the tonic signaling is CDR amino acids dependent, rather than to rescue these scFvs. In addition, we have now performed experiments to determine how alanine mutation will affect binding to IL13Rα2 and thus affect the function of CAR-Ts. New experiment data has been presented in the revised manuscript in Supplementary Figure S7. In one case, when mutating CDR3 of the light chain for CAR [B], named [B]-L3, we observed reduced tonic signaling with maintained binding to IL13Rα2. This was however not the case when simultaneously mutating CDR3 of the light chain and CDR2 of the heavy chain for CAR [B], named